Administration regime

ABSTRACT

A method for determining a folate substance administration regime is disclosed. The method comprises: quantifying, in a sample drawn from a patient, the expression level of at least one of the genes SLC46A1, SLC19A1, FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC; and establishing whether the expression level is high or low. A high expression level of at least one of said genes determines that said folate substance administration regime involves the administration of [6R]-methylenetetrahydrofolate and/or a folate substance upstreams of [6R]-methylenetetrahydrofolate in the metabolic pathway. A low expression level of at least one of said genes determines that said folate substance administration regime involves the administration of [6R]-methylenetetrahydrofolate. Also disclosed is a kit for determining such a folate substance administration regime.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of Ser. No. 16/601,306 filedon Oct. 14, 2019, which is a divisional of U.S. patent application Ser.No. 15/114,690 filed on Jul. 27, 2016, which is a U.S. national stagefiling under 35 U.S.C. § 371 of International Application No.PCT/EP2015/051947 filed on 30 Jan. 2015, which claims priority toEuropean Patent Application No. 14153538.5 filed on 31 Jan. 2014. Theentire disclosures of each of the above recited applications areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for determining a folatesubstance administration regime, especially as part of a cancertreatment.

BACKGROUND OF THE INVENTION

Folic acid and various kinds of folic acid derivatives are commonly usedas parts of different cancer treatments.

For example, leucovorin (folinic acid, LV) or its levo-isomer,levoleucovorin, and also other reduced folates, are frequently used incombination with fluorouracil (5-FU) in order to increase theanti-tumoral effect in treatment of patients with colorectal cancer.5-FU+LV (FLV) treatment may be used alone or in combination withoxaliplatin or irinotecan as adjuvant as well as palliative treatment ofcolorectal cancer. Leucovorin or levoleucovorin is also used incombination with methotrexate as a “rescue agent” in order to reduceside effects of the methotrexate.

[6R]-5,10-methylenetetrahydrofolate (6R-methylenetetrahydrofolate,[6R]-MTHF, Modufolin®) is an endogenous folate metabolite now beingdeveloped for direct administration to patients with the aim to increasethe efficacy and decrease the side effects of chemotherapeutic agentsused in the treatment of solid tumors. It has been suggested that theuse of the endogenous folate [6R]-MTHF in cancer treatment is morefavourable than the use of other folic acid derivatives, e.g.leucovorin, since [6R]-MTHF is the active agent resulting from folatemetabolism. In particular, it has been suggested that the administrationof e.g. leucovorin is less efficient than the administration of[6R]-MTHF, since many metabolic steps are required in order to achievethe active species, i.e. [6R]-MTHF, from leucovorin. In practice, it hasbeen seen that some patients nevertheless benefit from theadministration of leucovorin as a part of cancer treatment, while inother patients, the positive effects can hardly be seen at all. The sameis true for e.g. folic acid and levoleucovorin.

To date, the mechanisms underlying a successful folate treatment as partof a cancer treatment have not been resolved. The routine treatmentsinvolving e.g. folic acid, levoleucovorin or leucovorin are thereforefrequently used for all patients, although this means that thesesubstances are sometimes administered to patients as part of a cancertreatment without giving rise to any positive effects. This is notsatisfactory in terms of any risks associated with the administration ofunnecessary, non-active substances to patients, and also not in terms ofcost efficiency. Furthermore, it is not satisfactory that the positivesynergistic effects of the folate treatment, e.g. the increasedanti-tumoral effect, do not appear in some patients. Neither is itsatisfactory that the mediation of toxicity is difficult to predict orexpect with certainty. There is thus a need in the art for a morepredictable use of folic acid and folic acid derivatives in cancertreatment.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows estimated survival functions by level of SLC46A1 geneexpression in colorectal cancer tumor.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining a folatesubstance administration regime, said method comprising:

-   -   quantifying, in a sample drawn from a patient, the expression        level of at least one of the genes SLC46A1, SLC19A1, FPGS,        ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC; and    -   establishing whether said expression level is high or low,    -   wherein a high expression level of at least one of said genes        determines that said folate substance administration regime        involves the administration of [6R]-methylenetetrahydrofolate        and/or a folate substance upstreams of        [6R]-methylenetetrahydrofolate in the metabolic pathway, and        wherein a low expression level of at least one of said genes        determines that said folate substance administration regime        involves the administration of [6R]-methylenetetrahydrofolate.

In one embodiment of the present invention, the step of quantifying theexpression level of at least one of said genes is performed by real-timequantitative PCR, and the quantified expression level for each of saidgenes is preferably expressed as the Ct value normalized to the mean ofthe Ct values for the endogenous house-keeping genes β-actin and GAPDH.This normalized Ct value is also referred to as the ΔCt value.

In one embodiment of the present invention, the step of establishingwhether said expression level is high or low comprises comparing theexpression level of at least one of said genes with a predeterminedcutoff value. Suitable cutoff values have been experimentallyestablished, and the optimal cutoff values for available data are asfollows:

-   -   the predetermined cutoff value for SLC46A1 is 9.8;    -   the predetermined cutoff value for SLC19A1 is 7.8;    -   the predetermined cutoff value for FPGS is 5.5;    -   the predetermined cutoff value for ABCC3 is 6.6;    -   the predetermined cutoff value for MTHFD1L is 6.3;    -   the predetermined cutoff value for GGH is 6.2;    -   the predetermined cutoff value for MTHFD1 is 6.9;    -   the predetermined cutoff value for MTFMT is 7.2; and    -   the predetermined cutoff value for ATIC is 6.0;

Each predetermined cutoff value is expressed as the Ct value normalizedto the mean of the Ct values for the endogenous house-keeping genesβ-actin and GAPDH.

As will be explained further below, the mean of the Ct values for theendogenous house-keeping genes β-actin and GAPDH have been subtractedfrom the Ct values, so an increase in the normalized values represent adecrease in expression. Consequently, a high expression level isindicated by a quantified expression level below the respectivepredetermined cutoff value, whereas a low expression level is indicatedby a quantified expression level above the respective predeterminedcutoff value.

The folate substance upstreams of [6R]-methylenetetrahydrofolate in themetabolic pathway is suitably selected from the group consisting offolic acid (pteroyl-L-glutamic acid), mefolinate([6R,S]-5-CH₃-tetrahydrofolate), Metafolin®([6S]-5-CH₃-tetrahydrofolate), CoFactor®([6R,S]-5-CH₂-tetrahydrofolate), leucovorin([6R,S]-5-CHO-tetrahydrofolate), and/or levoleucovorin([6S]-5-CHO-tetrahydrofolate). In a preferred embodiment of theinvention, the folate substance upstreams of[6R]-methylenetetrahydrofolate is levoleucovorin or leucovorin.Furthermore, the folate substance administration regime is suitably partof a cancer treatment.

The present invention for the first time presents a way to provide acustomized folate treatment, especially as part of cancer treatment. Inparticular, the present invention has solved the problem of how topredict which patients who will benefit from a folate treatmentinvolving folic acid or folic acid derivatives, like leucovorin, andalso which patients who are more suited to be administered[6R]-methylenetetrahydrofolate directly. These findings are remarkable,and will provide a much more efficient and predictable handling offolate supplementation in cancer treatment, both from a medical and aneconomic perspective.

The invention is based on the surprising insight that the expressionlevel of certain genes has a direct effect on a patient's capability tometabolize folic acid and its derivatives into[6R]-methylenetetrahydrofolate. The identified genes belong to threedifferent categories:

-   -   folate transport (SLC46A1, SLC19A1, and ABCC3),    -   folate polyglutamation (FPGS and GGH), and    -   folate metabolism (MTHFD1L, MTHFD1, MTFMT, and ATIC)

According to one embodiment, the method according to the inventioninvolves quantifying the expression of at least one of the genesbelonging to the folate transport group, i.e. SLC46A1, SLC19A1, andABCC3.

According to another embodiment, the method according to the inventioninvolves quantifying the expression of at least one of the genesbelonging to the folate glutamation group, i.e. FPGS, and GGH.

According to a further embodiment, the method according to the inventioninvolves quantifying the expression of at least one of the genesbelonging to the folate metabolism group, i.e. MTHFD1 L, MTHFD1, MTFMT,and ATIC.

According to a preferred embodiment the method according to theinvention involves quantifying the expression of at least one gene fromeach group, i.e. at least one of the genes SLC46A1, SLC19A1, and ABCC3;at least one of the genes FPGS, and GGH; and at least one of the genesMTHFD1L, MTHFD1, MTFMT, and ATIC.

When the folate substance administration regime is used as part of acancer treatment, the cancer may be selected from the group consistingof breast cancer, gastric cancer, gall bladder cancer, bile duct cancer,colon cancer, rectal cancer, liver cancer, pancreatic cancer, head andneck cancer, and mesotheolioma cancer. The cancer treatment may involvethe administration of an anti-cancer agent selected from the groupconsisting of antifolates, anthracyclines, platinum derivatives,topoisomerase inhibitors, and antimetabolites, such as fluorouracil.

The sample to be analyzed according to the inventive method is suitablydrawn from a tumor of said patient, preferably a solid tumor.

The present invention also relates to a kit for determining a folatesubstance administration regime, said kit comprising:

-   -   means for quantifying, in a sample drawn from a patient, an        expression level of at least one of the genes SLC46A1, SLC19A1,        FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC; and    -   means for establishing whether said expression level is high or        low,    -   wherein a high expression level of at least one of said genes        determines that said folate substance administration regime        involves the administration of [6R]-methylenetetrahydrofolate        and/or a folate substance upstreams of        [6R]-methylenetetrahydrofolate in the metabolic pathway, and    -   wherein a low expression level of at least one of said genes        determines that said folate substance administration regime        involves the administration of [6R]-methylenetetrahydrofolate.

Based on the same inventive concept, the present invention also relatesto a method for identifying a patient susceptible for a folate substanceadministration regime involving the administration of a folate substanceupstreams of [6R]-methylenetetrahydrofolate in the metabolic pathway, aswell as a corresponding kit.

-   -   a method for identifying a patient particularly suited for a        folate substance administration regime involving the        administration of [6R]-methylenetetrahydrofolate, as well as a        corresponding kit.    -   a method for identifying a patient susceptible for a cancer        treatment involving the administration of an anti-cancer agent        (e.g. 5-FU) and a folate substance upstreams of [6R]-MTHF in the        metabolic pathway (e.g. leucovorin), as well as a corresponding        kit.    -   a method for identifying a patient who is not susceptible for a        cancer treatment involving the administration of an anti-cancer        agent (e.g. 5-FU) and a folate substance upstreams of [6R]-MTHF        in the metabolic pathway (e.g. leucovorin), as well as a        corresponding kit.    -   a method for identifying a patient particularly suited for a        cancer treatment involving the administration of an anti-cancer        agent (e.g. 5-FU) and [6R]-methylenetetrahydrofolate, as well as        a corresponding kit.

The inventive concept of identifying specific patient groups beingsusceptible for specific folate substance treatments also provides forestablishing new further medical uses of folate substances upstreams of[6R]-MTHF in the metabolic pathway, [6R]-MTHF, and anti-cancer agents.In particular, the present invention provides for

-   -   an anti-cancer agent and a folate substance upstreams of        [6R]-methylenetetrahydrofolate in the metabolic pathway for use        in the treatment of cancer in a patient showing a high        expression level of at least one of the genes SLC46A1, SLC19A1,        FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC.    -   an anti-cancer agent and [6R]-methylenetetrahydrofolate for use        in the treatment of cancer in a patient showing a low expression        level of at least one of the genes SLC46A1, SLC19A1, FPGS,        ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC.

Analogously, the present invention provides for:

-   -   a method for treatment of cancer in a patient showing a high        expression level of at least one of the genes SLC46A1, SLC19A1,        FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC, comprising        the administration of a pharmaceutically active amount of an        anti-cancer agent and a folate substance upstreams of        [6R]-methylenetetrahydrofolate in the metabolic pathway, and    -   a method for treatment of cancer in a patient showing a low        expression level of at least one of the genes SLC46A1, SLC19A1,        FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC, comprising        the administration of a pharmaceutically active amount of an        anti-cancer agent and [6R]-methylenetetrahydrofolate.

DETAILED DESCRIPTION OF THE INVENTION

In the research work leading to the present invention, it wassurprisingly found that high expression of certain folate-associatedgenes is associated with a better effect of leucovorin administrated aspart of cancer treatment. In particular, in a retrospective study inpatients with colorectal cancer, high expression of certainfolate-associated genes in tumor tissue was shown to correlate withdecreased risk of recurrent disease. Thus, it has been established thata low response to leucovorin therapy is linked to a low expression ofone or more of the identified folate-associated genes. Accordingly, thereason why some patients do not benefit from the administration ofleucovorin as a part of cancer treatment is that they are not able tometabolize the leucovorin into the active folate [6R]-MTHF to asufficient extent.

Patients showing a low expression of these genes therefore benefit fromthe administration of the directly active folate [6R]-MTHF, which doesnot require metabolic activation and is therefore independent of thesefolate-associated genes. The same concept applies for other folatesubstances upstreams of [6R]-MTHF in the metabolic cycle, since theyalso require metabolic activation to exert the desired effect. Allembodiments of the present invention are based on this surprisinginsight.

On a general level, the present invention for the first time providesfor the possibility to establish which patients that would benefit fromthe treatment of e.g. folic acid or leucovorin, and also which patientthat would not benefit from such a treatment. This involves a remarkableimprovement of the use of leucovorin and other folate substances incancer treatments, since based on these findings, tailor-made treatmentregimes can be established in order to provide as efficient treatmentsas possible. In particular, patients that do not have the capability tometabolize leucovorin are suitably administered the active folatemetabolite, [6R]-MTHF, directly.

The genes identified are SLC46A1; SLC19A1; FPGS; ABCC3; MTHFD1L; GGH;MTHFD1; MTFMT; and ATIC. All these genes belong to the state of the art,and their sequences are well-characterized. In accordance with thepresent invention, each of these genes are described by reference totheir respective reference human mRNA sequence(s) as provided by NCBI(National Center for Biotechnology Information).

In the NCBI database, the sequences are provided on the followingformat: [NM_X_(n).Y], wherein “NM” indicates an mRNA sequence; “X_(n)”indicates the accession number; and “Y” indicates the version number.The version numbers are continually updated, and in Table 1, the mostrecent version by the indicated date is provided.

The genes SLC46A1; SLC19A1; FPGS; ABCC3; MTHFD1L; GGH; MTHFD1; MTFMT;and ATIC to be quantified in accordance with the present invention arecompletely and unambiguously identified by the accession numbers andversion numbers indicated in Table 1 below. However, it is to beunderstood that similar reference sequences may also have the ability toprovide an accurate identification. In particular, other versions of thereference sequences as provided by NCBI also have the ability to providean accurate identification.

TABLE 1 Reference human Version mRNA sequence(s) number (NCBI Referenceas of 28 Transcript Sequence(s))- Base Jan Gene variant Accession numberpairs 2014 SLC46A1 1 NM_080669 6510 5 2 NM_001242366 6426 2 SLC19A1 1NM_194255 2873 2 2 NM_001205206 1885 1 3 NM_001205207 2622 1 FPGS 1NM_004957 2308 5 2 NM_001018078 2327 2 3 NM_001288803 2230 1 ABCC3 1NM_003786 5183 3 2 NM_001144070 1958 1 MTHFD1L 1 NM_001242767 3490 1 2NM_015440 3487 4 3 NM_001242768 3174 1 4 NM_001242769 1072 1 GGHNM_003878 1505 2 MTHFD1 NM_005956 3466 3 MTFMT NM_139242 2763 3 ATICNM_004044 2094 6

The detailed sequence information on each gene can be found on NCBI'sweb site: http://www.ncbi.nlm.nih.gov/refseq.

The reason why some of the genes are described by reference to more thanone mRNA reference sequence is that there exist mRNA splice variants.“Splicing” is a process occurring during gene expression, which resultsin a single gene coding for multiple proteins. In this process,particular exons of a gene may be included within, or excluded from, themRNA produced from that gene, i.e. the gene may give rise to severaldifferent mRNA splice variants.

Three of the identified genes are involved in folate transport (SLC46A1,SLC19A1, and ABCC3), two of the identified genes are involved in folatepolyglutamation (FPGS and GGH) and four of the identified genes areinvolved in folate metabolism (MTHFD1L, MTHFD1, MTFMT, and ATIC).

The gene SLC46A1 may alternatively be referred to as G21, HCP1, or PCFT.

The gene SLC19A1 may alternatively be referred to as CHMD, FOLT, IFC1,REFC, or RFC1.

The gene FPGS may alternatively be referred to as RP11-228B15.1.

The gene ABCC3 may alternatively be referred to as ABC31, EST90757,MLP2, MOAT-D, MRP3, or cMOAT2.

The gene MTHFD1L may alternatively be referred to as FTHFSDC1, MTC1THFS,RP1-292B18.2, or dJ292B18.2.

The gene GGH may alternatively be referred to as GH.

The gene MTHFD1 may alternatively be referred to as MTHFC, or MTHFD.

The gene MTFMT may alternatively be referred to as COXPD15, or FMT1.

The gene ATIC may alternatively be referred to as AICAR, AICARFT,IMPCHASE, OK/SW-c1.86, or PURH.

In its broadest sense, the present invention relates to a method fordetermining a folate substance administration regime, where theadministration regime is determined depending on the expression level ofone or more of the genes SLC46A1, SLC19A1, FPGS, ABCC3, MTHFD1L, GGH,MTHFD1, MTFMT, and ATIC.

If at least one of the genes SLC46A1, SLC19A1, FPGS, ABCC3, MTHFD1L,GGH, MTHFD1, MTFMT, and ATIC, shows a high expression level, it can bedetermined that the patient has the ability to metabolize a substanceupstreams of [6R]-MTHF in the metabolic pathway into [6R]-MTHF, andthus, the administration regime may involve the administration of e.g.leucovorin. Of course, this patient category also benefits from theadministration of [6R]-MTHF.

On the contrary, if at least one of the genes SLC46A1, SLC19A1, FPGS,ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC, shows a low expressionlevel, it can be concluded that the patient lacks the ability tosufficiently metabolize a substance upstreams of [6R]-MTHF in themetabolic pathway into [6R]-MTHF, and thus, the patient is suited for anadministration regime involving the administration of [6R]-MTHFdirectly.

By the expression “high expression level” is meant an expression levelof one of said genes, which involves a statistically significantdecreased risk of recurrent disease (colorectal cancer), measured asdisease-free survival, in patients treated with 5-FU in combination withleucovorin.

By the expression “low expression level” is meant an expression level ofone of said genes, which involves a statistically significant increasedrisk of recurrent disease (colorectal cancer), measured as disease-freesurvival, in patients treated with 5-FU in combination with leucovorin.

In the experimental part below, a detailed explanation on establishing ahigh and low expression level, respectively, is provided.

In order to determine whether a patient shows a high or a low expressionof a certain gene, the expression of one or more genes of interest issuitably examined by quantification by real-time quantitative PCR, whichis a well-established technique used in the field for analysing geneexpression levels. The quantified expression level for each of saidgenes is preferably expressed as the Ct value normalized to the mean ofthe Ct values for the endogenous house-keeping genes ACTB (β-actin) andGAPDH. These genes belong to the state of the art, and their sequencesare well-characterized.

In accordance with the present invention, each of these housekeepinggenes are described by reference to their respective reference humanmRNA sequence(s) as provided by NCBI (National Center for BiotechnologyInformation).

In the NCBI database, the sequences are provided on the followingformat: [NM_X_(n).Y], wherein “NM” indicates an mRNA sequence; “X_(n)”indicates the accession number; and “Y” indicates the version number.The version numbers are continually updated, and in Table 2, the mostrecent version by the indicated date is provided.

The house-keeping genes ACTB (β-actin) and GAPDH to be quantified inaccordance with the present invention are completely and unambiguouslyidentified by the accession numbers and version numbers indicated inTable 2 below. However, it is to be understood that similar referencesequences may also have the ability to provide an accurateidentification. In particular, other versions (i.e. reference sequenceshaving the same accession numbers, but different version numbers) of thereference sequences as provided by NCBI also have the ability to providean accurate identification.

TABLE 2 Reference human Version mRNA sequence(s) number House- (NCBIReference as of 28 keeping Transcript Sequence(s))- Base Jan genevariant Accession number pairs 2014 ACTB NM_001101 1852 3 GAPDH 1NM_002046 1421 5 2 NM_001256799 1455 2

The gene ACTB may alternatively be referred to as BRWS1, or PS1TP5BP1.

The gene GAPDH may alternatively be referred to as CDABP0047, G3PD, orGAPD.

In a real-time quantitative PCR assay a positive reaction is detected byaccumulation of a fluorescent signal. The Ct, “cycle threshold”, isdefined as the number of cycles required for the fluorescent signal tocross the threshold (i.e. exceeds background level). Ct levels areinversely proportional to the amount of target nucleic acid in thesample (i.e. the lower the Ct level the greater the amount of targetnucleic acid in the sample).

Normalization to an endogenous control (often referred to as ahousekeeping gene) allows correction of results that can otherwise beskewed by differing amounts of input nucleic acid template. Any geneshown to be expressed at the same level in all study samples canpotentially be used as an endogenous control.

According to the present invention, the step of establishing whether anexpression level is high or low suitably comprises comparing theexpression level of a gene of interest with a predetermined cutoffvalue. Suitable cutoff values have been experimentally established, andare listed in Table 6 below.

As the mean of the Ct values for the endogenous house-keeping genesβ-actin and GAPDH have been subtracted from the Ct values, an increasein the normalized values represent a decrease in expression.Consequently, a high expression level is indicated by a quantifiedexpression level below the respective predetermined cutoff value,whereas a low expression level is indicated by a quantified expressionlevel above the respective predetermined cutoff value.

Specific examples of assays that may be used for the analyzing theexpression of the genes according to the invention are exemplified inTables 3 and 4 (see also

Example 1 and Table 5 below (“Assay ID”)). The assays are provided by“Life Technologies”, and detailed information on each assay can be foundon their website: http://www.lifetechnologies.com/.

As evidenced in Tables 3 and 4, the reference human mRNA sequencesincluded in the exemplified analyses do not always have the versionnumbers listed in Tables 1 and 2. The reason for this is that there theexemplified assays were developed at a point in time where a previousversion of the reference sequence was the most recent version.

TABLE 3 Reference human mRNA sequence(s) (NCBI Reference Sequence(s))Base Gene Assay ID included in analysis pairs SLC46A1 Hs00611081_m1NM_080669.5 6510 SLC19A1 Hs00953344_m1 NM_194255.2 2873 NM_001205206.11885 NM_001205207.1 2622 FPGS Hs00191956_m1 NM_004957.4 2487NM_001018078.1 2475 ABCC3 Hs00358656_m1 NM_003786.3 5183 NM_001144070.11958 MTHFD1L Hs00383616_m1 NM_001242767.1 3490 NM_015440.4 3487NM_001242768.1 3174 GGH Hs00914163_m1 NM_003878.2 1505 MTHFD1Hs00602830_m1 NM_005956.3 3466 MTFMT Hs00373739_m1 NM_139242.3 2763 ATICHs00269671_m1 NM_004044.6 2094

TABLE 4 Reference human mRNA sequence(s) House- (NCBI Reference keepingSequence(s)) Base gene Assay ID included in analysis pairs ACTBHs99999903_m1 NM_001101.3 1852 GAPDH Hs99999905_m1 NM_002046.4 1401

The gene expression is preferably analyzed in a tissue sample drawn froma tumor of a patient, typically by a biopsy.

In one embodiment of the invention, the sample is analyzed for theexpression of one or more genes from one of the gene groups mentionedabove (i.e. genes involved in folate transport, folate polyglutamation,and folate metabolism, respectively). In another embodiment the sampleis analyzed for the expression of one or more genes from two of thegroups. In a preferred embodiment, the sample is analyzed for theexpression of one or more genes from all of the three respective groups.In another preferred embodiment, the sample is analyzed for theexpression of all nine genes. In one embodiment, the sample is analyzedfor the expression of SLC46A1 and/or SLC19A1.

Some alternative denotations of [6R]-methylenetetrahydrofolate are:Modufolin®, [6R]-5,10- methylenetetrahydrofolate, [6R]-methylene-THF,[6R]-5,10-methylene-THF, [6R]-MTHF, [6R]-5,10-MTHF, [6R]-5,10-CH₂-THF,[6R]-5,10-CH₂-tetrahydrofolate, [6R]-5,10-CH₂-H₄PteGlu_(n), The IUPACname of methylenetetrahydrofolate is:N-[4-(3-amino-1-oxo-1,4,5,6,6a,7-hexahydroimidazo[1,5-f]pteridin-8(9H)-yl)benzoyl]-L-glutamicacid.

By the expression “a folate substance upstreams of [6R]-MTHF in themetabolic pathway” is meant any folate substance that uponadministration to a patent is metabolized in the body to [6R]-MTHF.Examples of such substances are folic acid (pteroyl-L-glutamic acid),mefolinate ([6R,S]-5-CH₃-tetrahydrofolate), Metafolin®([6S]-5-CH₃-tetrahydrofolate), CoFactor®([6R,S]-5-CH₂-tetrahydrofolate), leucovorin([6R,S]-5-CHO-tetrahydrofolate), and levoleucovorin([6S]-5-CHO-tetrahydrofolate). The expression “metabolic pathway” is arecognized expression within biochemistry and relates to a series ofchemical reactions in a cell, catalyzed by enzymes and connected bytheir intermediates, i.e. the reactants of one reaction are the productsof the previous one. The expression “upstreams of” defines that thefolate substance in question is involved in a chemical reaction whichoccurs at an earlier stage in the series of chemical reactions than thechemical reaction leading to [6R]-MTHF.

Although the invention refers to a general method for determining afolate substance administration regime, it is particularly suitable fordetermining a folate administration regime which is part of a cancertreatment.

The present invention may be used in connection to the treatment ofseveral cancer forms, e.g. breast cancer, gastric cancer, gall bladdercancer, bile duct cancer, colon cancer, rectal cancer, liver cancer,pancreatic cancer, head and neck cancer, and mesotheolioma cancer.

The cancer treatment may involve the administration of variousanti-cancer agents, like antifolates, anthracyclines, platinumderivatives, topoisomerase inhibitors, and antimetabolites. Theanti-cancer agents may be used alone or in combination. Suitableadministration regimes of anti-cancer agents are known to a personskilled in the art.

Examples of antifolates to be used in accordance with the presentinvention are pemetrexed (Alimta®), raltitrexed (Tomudex®),methotrexate, and pralatrexate, all of which are transported by RFC(Reduced Folate Carrier). Other antifolates of interest aretumor-targeted antifolates, e.g. non-benzoyl 6-substituted straightchain pyrrolo[2,3-d]pyrimidine antifolates, that are specific substratesfor transporters other than RFC with limited expression and/or transportinto normal tissues compared with tumors. Examples of anthracyclines tobe used in accordance with the present invention are daunorubicin,doxorubicin, epirubicin, idarubicin, and valrubicin. Examples ofplatinum derivatives to be used in accordance with the invention areoxaliplatin, cisplatin, carboplatin, satraplanin, picoplatin,nedaplatin, and triplatin. Examples of topoisomerase inhibitors to beused in accordance with the invention are irinotecan, topotecan, andcamptothecin. Examples of antimetabolites to be used in accordance withthe invention are fluorouracil (5-FU), floxuridine, cytosinearabinoside, and 6-azauracil.

The present invention also relates to a kit for determining a folateadministration regime as defined above. Such a kit comprises some kindof means for quantifying, in a sample drawn from a patient, theexpression level of at least one of the genes SLC46A1, SLC19A1, FPGS,ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC.

The means may comprise an assay for analyzing the expression of one ormore genes from one of the gene groups mentioned above (i.e. genesinvolved in folate transport (SLC46A1, SLC19A1, and ABCC3), folatepolyglutamation (FPGS and GGH), and folate metabolism (MTHFD1L, MTHFD1,MTFMT, and ATIC), respectively). In another embodiment the meanscomprises an assay for analyzing the expression of one or more genesfrom two of the groups. In a preferred embodiment, the means comprisesan assay for analyzing the expression of one or more genes from all ofthe three respective groups. In another preferred embodiment, the meanscomprises an assay for analyzing the expression of each of the ninegenes (SLC46A1, SLC19A1, ABCC3, FPGS, GGH, MTHFD1L, MTHFD1, MTFMT, andATIC). In one embodiment, the means comprises an assay for analyzing theexpression of SLC46A1 and/or SLC19A1.

Suitably, the kit also comprises a leaflet indicating that a highexpression level of at least one of the genes SLC46A1, SLC19A1, FPGS,ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC, determines that thepatient from which the sample is drawn is suited for a folate substanceadministration regime involving the administration of [6R]-MTHF and/or afolate substance upstreams of [6R]-MTHF in the metabolic pathway, andfurther that a low expression level of at least one of said genesSLC46A1, SLC19A1, FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC,determines that the patient from which the sample is drawn is suited fora folate substance administration regime involving the administration of[6R]-MTHF.

Based on the same inventive concept, the invention also relates to amethod for identifying a patient susceptible for a folate substanceadministration regime involving the administration of a folate substanceupstreams of [6R]-MTHF in the metabolic pathway, as well as to acorresponding kit.

Based on the same inventive concept, the invention also relates to amethod for identifying a patient particularly suited for a folatesubstance administration regime involving the administration of[6R]-MTHF, as well as to a corresponding kit.

The underlying inventive concept also provides for a method foridentifying a patient susceptible for a cancer treatment involving theadministration of an anti-cancer agent (e.g. 5-FU) and a folatesubstance upstreams of [6R]-MTHF in the metabolic pathway (e.g.leucovorin), as well as a corresponding kit.

The underlying inventive concept also provides for a method foridentifying a patient who is not susceptible for a cancer treatmentinvolving the administration of an anti-cancer agent (e.g. 5-FU) and afolate substance upstreams of [6R]-MTHF in the metabolic pathway (e.g.leucovorin), as well as a corresponding kit.

The underlying inventive concept also provides for a method foridentifying a patient particularly suited for a cancer treatmentinvolving the administration of an anti-cancer agent (e.g. 5-FU) and[6R]-methylenetetrahydrofolate, as well as a corresponding kit.

The inventive concept of identifying specific patient groups beingsusceptible for specific folate substance treatments also provides forestablishing new further medical uses of folate substances upstreams of[6R]-MTHF in the metabolic pathway, [6R]-MTHF, and anti-cancer agents.

In particular, the present invention provides for an anti-cancer agent(e.g. 5-FU) and a folate substance upstreams of [6R]-MTHF in themetabolic pathway (e.g. leucovorin) for use in the treatment of cancerin a patient showing a high expression level of at least one of thegenes SLC46A1, SLC19A1, FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, andATIC. The present invention also provides for an anti-cancer agent (e.g.5-FU) and [6R]-MTHF for use in the treatment of cancer in a patientshowing a low expression level of at least one of the genes SLC46A1,SLC19A1, FPGS, ABCC3, MTHFD1L, GGH, MTHFD1, MTFMT, and ATIC.

The invention will now be further explained in the following examples.These examples are only intended to illustrate the invention and shouldin no way be considered to limit the scope of the invention.

EXAMPLES Example 1

The aim of Example 1 was to identify genes in the folate pathway withpossible impact on the metabolism of LV, given according to the Nordicbolus regime (Carlsson, et al.; Sequential 5-fluorouracil and leucovorinin patients with advanced symptomatic gastrointestinal cancer, Eur JCancer 1990;26:874-6). Relative expression of 22 genes putativelyinvolved in transport, polyglutamation and metabolism of LV wasdetermined and related to disease-free survival (DFS) of patients withstage III colorectal cancer, who were given adjuvant treatment with FLVor with 5-FU, LV and oxaliplatin (FLOX).

Patients and Methods Patients

During the period 2001-2009, 542 patients with non-hereditary stage IIIcolorectal cancer underwent surgery at the Sahlgrenska UniversityHospital/Östra and received adjuvant FLV (n=383) or FLOX (n=159)treatment. Out of the 542 patients, 446 underwent elective surgery. Toenable comparison of gene expression in matched tumor and mucosasamples, it was necessary to collect and snap-freeze biopsies atsurgery. Biopsy samples were obtained from 290 patients, and of these193 were available for the current study.

All tumors were classified according to the Tumor-Node-Metastasis (TNM)staging system (Compton C, et al.; American Joint Committee on CancerPrognostic Factors Consensus Conference: Colorectal Working Group;Cancer 2000;88:1739-57). Patients were followed for 3-5 years aftersurgical removal of the primary tumor. The ethics committee of theUniversity of Gothenburg approved the study and informed consent wasobtained from all patients.

Treatment

Patients received adjuvant treatment with FLV or FLOX. FLV treatmentcomprised intravenous bolus injections of 5-FU (500 mg/m²), followed byLV (60 mg/m²) 30-40 minutes later, once weekly or on Days 1 and 2 everyother week, according to the Nordic FLV regime (Carlsson , et al.;Sequential 5-fluorouracil and leucovorin in patients with advancedsymptomatic gastrointestinal cancer, Eur J Cancer 1990;26:874-6). FLOXtreatment comprised 5-FU (500 mg/m²) and LV (60 mg/m²) bolus on Days 1and 2 every other week, plus an oxaliplatin infusion (85 mg/m²) over 120minutes on Day 1 every other week (Sorbye H, Dahl O; Nordic5-fluorouracil/leucovorin bolus schedule combined with oxaliplatin(Nordic FLOX) as first-line treatment of metastatic colorectal cancer,Acta Oncol 2003;42:827-31).

Selection of Genes

Based on recent scientific literature (Sadahiro S, et al., Moleculardeterminants of folate levels after leucovorin administration incolorectal cancer, Cancer Chemother Pharmacol 2010;65:735-42.), 22target genes with putative impact on LV metabolism were chosen foranalysis (Table 5). Seven of these genes are involved in folatetransport (ABCC1, ABCC3, FOLR1, FOLR2, FOLR3, SLC19A1/RFC-1 andSLC46A1/PCFT), whereas the other 15 genes encode enzymes involved infolate polyglutamation (FPGS and GGH) or folate metabolism (ALDH1L1,ALDH1L2, AMT, ATIC, FTCD, GART, MTFMT, MTHFD1, MTHFD1L, MTHFD2, MTHFS,SHMT1 and SHMT2).

TABLE 5 Gene list Gene Gene category symbol Gene name Assay ID FolateABCC1 ATP-binding cassette, sub-family C (CFTR/MRP), member 1Hs00219905_m1 transport ABCC3 ATP-binding cassette, sub-family C(CFTR/MRP), member 3 Hs00358656_m1 FOLR1 Folate receptor 1 (adult)Hs01124177_m1 FOLR2 Folate receptor 2 (fetal) Hs00265255_m1 FOLR3 Folatereceptor 3 (gamma) Hs01549264_m1 SLC19A1 Solute carrier family 19(folate transporter), member 1 Hs00953344_m1 SLC46A1 Solute carrierfamily 46 (folate transporter), member 1 Hs00611081_m1 Folate ALDH1L1Aldehyde dehydrogenase 1 family, member L1 Hs00201836_m1 metabolismALDH1L2 Aldehyde dehydrogenase 1 family, member L2 Hs00402876_m1 AMTAminomethyltransferase Hs00166628_m1 ATIC 5-Aminoimidazole-4-carboxamideribonucleotide formyltransferase/IMP Hs00269671_m1 cyclohydrolase FTCDFormiminotransferase cyclodeaminase HS00198409_m1 GARTPhosphoribosylglycinamide formyltransferase, phosphoribosylglycinamideHs00531926_m1 synthetase, phosphoribosylaminoimidazole synthetase MTFMTMitochondrial methionyl-tRNA formyltransferase Hs00373739_m1 MTHFD1Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1,Hs00602830_m1 methenyltetrahydrofolate cyclohydrolase,formyltetrahydrofolate synthetase MTHFD1L Methylenetetrahydrofolatedehydrogenase (NADP+ dependent) 1-like Hs00383616_m1 MTHFD2Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2,Hs00741165_m1 methenyltetrahydrofolate cyclohydrolase MTHFS5,10-methenyltetrahydrofolate synthetase (5-formyltetrahydrofolateHs00197574_m1 cyclo-ligase) SHMT1 Serine hydroxymethyltransferase 1(soluble) Hs00541038_m1 SHMT2 Serine hydroxymethyltransferase 2(mitochondrial) Hs00193658_m1 Folate FPGS Folylpolyglutamate synthaseHs00191956_m1 poly- GGH Gamma-glutamyl hydrolase (conjugase,Hs00914163_m1 glutamation folylpolygammaglutamyl hydrolase) House- ACTBActin, beta Hs99999903_m1 keeping GAPDH Glyceraldehyde-3-phosphatedehydrogenase Hs99999905_m1Preparation of RNA and cDNA

Tumor and matched macroscopically normal-appearing mucosa (obtainedapproximately 10 cm from the tumor) were snap-frozen in liquid nitrogenafter removal and stored at −70° C. until used. Total RNA was isolatedfrom 10-30 mg fresh-frozen tissue using the High Pure RNA Tissue Kit (#12033674001, Roche Diagnostics Scandinavia AB) according to themanufacturer's instructions. cDNA was synthesized using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems) and run onGene Amp PCR System 9600 (Perkin Elmer). To optimize each run, theexpression level of β-actin was determined in each sample. A second RNAextraction and cDNA synthesis were performed if the concentration wasconsidered to be suboptimal.

Real-Time Quantitative PCR

The relative gene expression was quantified using TaqMan® Low-DensityArray (TLDA) cards (Applied Biosystems). Custom-designed TLDA cardscontaining 24 individual assays were ordered from Applied Biosystems athttp://www.appliedbiosystems.com. Three samples and one calibrator(SK-N-AS) were loaded to each card according to the manufacturer'sinstructions; each reservoir contained 83 ng of RNA converted to cDNA ina total volume of 100 μl. Two test runs were performed before the actualanalysis. Quantitative polymerase chain reactions (QPCRs) were set up induplicates in 384-well plates using the Biomek FX pipetting robot(Beckman Coulter) and were carried out in 10 μl reactions with 1×TaqMan®Gene Expression Mastermix (Applied Biosystems), 1×gene-specific assayand 7.5 ng RNA converted into cDNA. Both TLDA cards and individual QPCRplates were run and analyzed by the ABI PRISM® 7900HT Sequence DetectionSystem (SDS 2.2, Applied Biosystems) according to the manufacturer'sprotocol. Calculations were performed using the ΔΔCt relativequantification method. The thresholds and baselines were set manually inSDS and Ct values were extracted. All Ct values were normalized to themean of the endogenous house-keeping genes β-actin and GAPDH for eachsample.

Statistics

Statistical analysis was performed using the ‘survival’ package in the Rstatistical software (Therneau T; A Package for Survival Analysis in S.R package version 2.37-4, http://CRAN.R-project.org/package=survival;2013). Cox proportional-hazards regression models were applied to thedata to examine the relationship between expression levels of chosengenes and the different censor variables. To choose between the numerousclinical covariates, stepwise model selection by Akaike informationcriterion (AIC) was performed on Cox models excluding the expressionvalues. AIC is a measure of relative goodness of fit and, as long as itimproved the AIC-value, the covariate that gave the best AIC if removedwas deleted. The selected covariates were then included in the finalmodels.

Cox regression was performed individually for each gene, for each genefor expression values from mucosa and tumor separately, and for onecensor variable at a time. Furthermore, the difference between themucosa and tumor was investigated in a separate model for each gene.Each of these models was fitted both with and without the additionalselected clinical covariates to examine their influence on the models.Additionally, each pair of genes (tumor and mucosa) was fitted togetherto a Cox model in the same fashion, to examine possible interactions.

Statistical values of p≤0.05 were judged to be significant. Permutationswere used to correct p-values for multiple testing. Furthermore, usingthe “qvalue” package in the R statistical software, false discovery rateq-values were calculated (Dabney A and Storey J D with assistance fromWarnes G R; qvalue: Q-value estimation for false discovery rate control;R package version 1.32.0). There was a linear correlation between thetwo house-keeping genes β-actin and GAPDH. The expression of the targetgenes was related to a mean value representing both of these genes inorder to keep variance to the minimum. DFS was calculated from the dateof surgery to the last follow-up, or to the date of recurrence or death.All subjects that were disease-free at five years were censored at fiveyears.

Determination of Cutoff Values

The optimal cutoff values were derived for one gene at the time. Foreach gene all possible cutoff values where none of the two groupsconsisted of less than 25% of the total number of observations weretested. Each cutoff value was tested through a logrank test comparingsurvival distributions of the two groups, and the cutoff value resultingin the smallest p-value from the logrank test was selected.

Calculations are made on Ct values normalized to the mean of theendogenous house-keeping genes β-actin and GAPDH for each sample. Themeans of the house-keeping genes were subtracted from the Ct values, soan increase in the normalized values still represents a decrease inexpression.

The resulting cutoff values are presented in Table 6.

TABLE 6 Gene CutOff (ΔCt) ABCC3 6.6 ATIC 6.0 FPGS 5.5 GGH 6.2 MTFMT 7.2MTHFD1 6.9 MTHFD1L 6.3 SLC19A1 7.8 SLC46A1 9.8

Results

Patient and tumor characteristics are shown in Table 7. The median ageof patients at surgery was 65 years (range 35-80) and 92 (48%) patientswere female. All tumors were classified as TNM stage III.

TABLE 7 Patients (n = 193) and tumor characteristics Gender, n (%)Female  92 (48) Male 101 (52) Age, median (range)  65 (35-80) Tumorlocation, n (%) Right-sided colon  47 (24) Left-sided colon  56 (29)Right- and left-sided colon  2 (1) Rectum  88 (46) Tumordifferentiation, n (%) High/Moderate 152 (79) Low  32 (16) Unknown  9(5) Assessed lymph nodes,  18 (3-63) median (range) Positive lymphnodes,  2 (1-31) median (range) Lymph node ratio,  15 (1.6-86) median(range)

The multivariate Cox regression analysis included the parameters gender,age, lymph node ratio, tumor differentiation grade and adjuvant therapy(FLV or FLOX). Due to very low expression values of the house-keepinggenes, one patient was considered to be an outlier and was excluded fromthe Cox regression analysis. When doing stepwise variable selection withAIC, the two variables that emerged and which were included in the modelwere lymph node ratio and adjuvant therapy. Both varied to some extentin the different models, with lymph node ratio constantly having valuesof p<0.01, while adjuvant therapy was significant (at the p≤0.05 level)in only 42% of the models, although it constantly had values of p<0.1.

During follow-up, 59 of the 193 (31%) patients relapsed. A similarrecurrence rate was found in 92 of the 253 (36%) patients who underwentelective surgery but from whom biopsy samples were not available. Thisindicates that the selected patient group was representative of thetotal cohort of patients treated with adjuvant FLV or FLOX therapy in2001-2009.

There was a significant correlation between low expression of thefollowing genes in tumor tissue and a increased risk of recurrentdisease, measured as DFS: SLC46A1/PCFT (p<0.001), ABCC3 (p<0.01), FPGS(p<0.01), SLC19A1/RFC-1 (p<0.01), RTIC (p<0.05), GGH (p<0.05), MTFMT(p<0.05), MTHFD1 (p<0.05) and MTHFD1L (p<0.05) (Table 8). Aftercorrection for multiple testing, SLC46A1/PCFT and SLC19A1/RFC-1 remainedsignificant with p-values <0.05.

TABLE 8 Hazard Gene ratio^(a) P Pc^(b) q^(c) SLC46A1/PCFT 1.30 0.000890.021 0.012 SLC19A1/RFC-1 1.39 0.0016 0.035 0.012 FPGS 1.45 0.0038 0.0710.019 ABCC3 1.24 0.0051 0.093 0.019 MTHFD1L 1.30 0.016 0.24 0.049 GGH1.18 0.025 0.34 0.065 MTHFD1 1.32 0.038 0.45 0.070 MTFMT 1.38 0.038 0.460.070 ATIC 1.43 0.041 0.48 0.070 GART 1.16 0.17 0.92 0.25 ABCC1 1.170.18 0.93 0.25 SHMT1 0.92 0.26 0.98 0.31 ALDH1L2 1.11 0.26 0.98 0.31MTHFD2 0.92 0.38 1.0 0.42 FTCD 0.96 0.51 1.0 0.52 SHMT2 1.06 0.64 1.00.60 FOLR1 1.02 0.66 1.0 0.60 FOLR2 1.02 0.72 1.0 0.60 MTHFS 0.95 0.741.0 0.60 ALDH1L1 0.99 0.79 1.0 0.60 AMT 1.00 0.96 1.0 0.68 FOLR3 1.000.97 1.0 0.68 ^(a)Hazard ratios are calculated from ΔCt values andcorrespond to a halving of the expression levels. ^(b)p-values correctedfor multiple testing with permutations. ^(c)False discovery rateq-value.

Almost identical survival functions were obtained when the expressionlevels of the SLC46A1/PCFT, ABCC3, FPGS and SLC19A1/RFC-1 genes wereplotted against time. A representative survival curve is shown in FIG. 1. FIG. 1 shows the estimated survival functions by level of SLC46A1/PCFTgene expression in colorectal cancer tissue. The two curves showpredicted survival functions for the fitted Cox proportional hazardsmodels. The expression levels for the two curves have been set onestandard deviation above, and one standard deviation below the mean,where one standard deviation corresponds to a fold change in logexpression levels of 1.57. Lymph node ratio has been set to its mean,and adjuvant therapy has been set to FLV since it was the treatmentgiven to a majority of the patients. Standard deviations and mean forexpression levels have been calculated from ΔCt values.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent for one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A method for determining a treatment regime comprising administrationof a folate to a patient in need thereof, said method comprising: (a)quantifying, in a sample obtained from said patient, the expressionlevel of at least one gene selected from the group consisting ofSLC46A1, SLC19A1, FPGS, ABCC3, SHMT1, MTHFD1L, GGH, MTHFD1, MTFMT, andATIC; and (b) establishing whether said expression level is high or low,wherein a low expression level of at least one of said genes determinesthat said treatment regime will comprise the administration of[6R]-methylenetetrahydrofolate to a patient in need thereof; and whereina high expression level of at least one of said genes determines thatsaid treatment regime will comprise the administration of a folateselected from the group consisting of folic acid,[6R,S]-5-CH₃-tetrahydrofolate, [6S]5-CH₃-tetrahydrofolate,[6R,S]-5-CH₂-tetrahydrofolate, [6S]-5-CHO-tetrahydrofolate, and[6R,S]-5-CHO-tetrahydrofolate to a patient in need thereof.
 2. A methodaccording to claim 1, wherein the step of quantifying the expressionlevel of at least one of said genes is performed by real-timequantitative PCR.
 3. A method according to claim 1, wherein theexpression level of each of said genes is expressed as the Ct valuenormalized to the mean of the Ct values for an endogenous house-keepinggene selected from the group consisting of β-actin and GAPDH.
 4. Amethod according to claim 1, wherein the step of establishing whethersaid expression level is high or low comprises comparing the expressionlevel of at least one of said genes with a predetermined cutoff value.5. A method according to claim 4, wherein: the predetermined cutoffvalue for SLC46A1 is 9.8; the predetermined cutoff value for SLC19A1 is7.8; the predetermined cutoff value for FPGS is 5.5; the predeterminedcutoff value for ABCC3 is 6.6; the predetermined cutoff value forMTHFD1L is 6.3; the predetermined cutoff value for GGH is 6.2; thepredetermined cutoff value for MTHFD1 is 6.9; the predetermined cutoffvalue for MTFMT is 7.2; and the predetermined cutoff value for ATIC is6.0; wherein each predetermined cutoff value is expressed as a Ct valuenormalized to a mean Ct value for endogenous house-keeping genesselected from the group consisting of β-actin and GAPDH, and wherein ahigh expression level is indicated by an expression level below apredetermined cutoff value, and a low expression level is indicated byan expression level above a predetermined cutoff value.
 6. A methodaccording to claim 1, said method comprising quantifying the expressionlevel of at least one of the genes SLC46A1, SLC19A1, and ABCC3; at leastone of the genes FPGS, and GGH; or at least one of the genes MTHFD1L,MTHFD1, MTFMT, and ATIC.
 7. A method according to claim 1, wherein saidsample is obtained from a tumor of said patient.
 8. A method foridentifying a patient for a treatment regime, said treatment regimecomprising administration of [6R]-methylenetetrahydrofolate to saidpatient, said method comprising: (a) quantifying, in a sample obtainedfrom said patient, an expression level of at least one gene selectedfrom the group consisting of SLC46A1, SLC19A1, FPGS, ABCC3, SHMT1,MTHFD1L, GGH, MTHFD1 , MTFMT, and ATIC; and (b) establishing whethersaid expression level is high or low, wherein a low expression level ofat least one of said genes identifies said patient for administration of[6R]-methylenetetrahydrofolate.